Concurrent assessment of calpain and caspase-3 activation after oxygen-glucose deprivation in primary septo-hippocampal cultures

The contributions of calpain and caspase-3 to apoptosis and necrosis after central nervous system (CNS) trauma are relatively unexplored. No study has examined concurrent activation of calpain and caspase-3 in necrotic or apo ptotic cell death after any CNS insult. Experiments used a model of oxygen- glucose deprivation (OGD) in primary septo-hippocampal cultures and assesse d cell viability, occurrence of apoptotic and necrotic cell death phenotype s, and protease activation. Immunoblots using an antibody detecting calpain and caspase-3 proteolysis of alpha -spectrin showed greater accumulation o f calpain-mediated breakdown products (BDPs) compared with caspase-3-mediat ed BDPs. Administration of calpain and caspase-3 inhibitors confirmed that activation of these proteases contributed to cell death, as inferred by lac tate dehydrogenase. release. Oxygen-glucose deprivation resulted in express ion of apoptotic and necrotic cell death phenotypes, especially in neurons. Immunocytochemical studies of calpain and caspase-3 activation in apoptoti c cells indicated that these proteases are almost always concurrently activ ated during apoptosis. These data demonstrate that calpain and caspase-3 ac tivation is associated with expression of apoptotic cell death phenotypes a fter OGD, and that calpain activation, in combination with caspase-3 activa tion, could contribute to the expression of apoptotic cell death by assisti ng in the degradation of important cellular proteins.